Non alcoholic fatty liver disease (NAFLD) is the most common liver disease in the USA and may be present in up to 30% of the general population. A subset of patients with NAFLD has non-alcoholic steatohepatitis (NASH), a more severe form of this disease associated with hepatocellular injury, inflammation and varying levels of fibrosis. Patients with NASH are at increased risk of progression to cirrhosis and end-stage liver disease. It is estimated that about 6 million Americans are at risk of developing cirrhosis due to NAFLD. Thus, there is an urgent need to understand the mechanisms whereby patients with NAFLD develop NASH. The "two-hit" hypothesis suggests that the primary insult in NAFLD is insulin resistance and increased circulating free fatty acid levels;NASH may develop in the presence of a "second hit" leading to increased oxidative stress in the liver. A large body of laboratory and clinical data suggests that hepatic iron overload results in production of reactive oxygen species, which may lead to increased liver injury and hepatic fibrogenesis via induction of a number of pathways, many of which involve the redox-sensitive nuclear factor-:B (NF-:B). In a preliminary study of more than 800 subjects enrolled in the NASH Clinical Research Network (CRN), we have found that hepatic iron deposition is present in 35% of NAFLD patients, or an estimated 15 million adult Americans, and that the presence of hepatic iron deposition in reticuloendothelial cells is associated with NASH and advanced fibrosis. By contrast, hepatocellular iron deposition is associated with less severe histologic disease. We are proposing a systems biology approach to identify genomic variants, gene expression profiles and serum and immunohistochemical biomarkers which may explain these dichotomous hepatic iron phenotypes. The proposed studies are feasible, will harness the vast resources of the NASH CRN and will provide an extraordinarily unique opportunity to determine the pathways and mechanisms involved in the pathogenesis of NASH, including but not limited to role of hepatic iron. We believe our results could lead to a greater understanding of the mechanism causing this common but poorly understood liver disease and may lead to novel therapeutic interventions. PUBLIC HEALTH RELEVANCE: NAFLD is the most common liver disease in Western societies. It is estimated that about 6 million Americans are at risk of developing cirrhosis from nonalcoholic fatty liver disease (NAFLD). We have identified that more than one third of patients with NAFLD have hepatic iron deposition (an estimated 15 million adult Americans) which may contribute to disease progression. The goal of this study is to determine the mechanisms leading to hepatic iron deposition and more severe NAFLD.